Formulations and methods for contemporaneous stabilization of active proteins during spray drying and storage

ABSTRACT

A method of treatment of plasma with a physiologically compatible spray dry stable acidic substance (SDSAS) prior to or contemporaneously with spray drying of the plasma that results in greater recovery and greater long-term stabilization of the dried plasma proteins as compared to spray dried plasma that has not be subject to the formulation method of the present invention, as well as compositions related to plasma dried by the methods of the present invention.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/383,201, entitled, “Formulations And Methods For ContemporaneousStabilization Of Active Proteins During Spray Drying And Storage” byQiyong Peter Liu et al., filed Dec. 19, 2016, which is a continuation ofU.S. application Ser. No. 14/858,539, now U.S. Pat. No. 9,545,379,entitled, “Formulations And Methods For Contemporaneous Stabilization OfActive Proteins During Spray Drying And Storage” by Qiyong Peter Liu etal., filed Sep. 18, 2015, issued Jan. 17, 2017, which claims the benefitof U.S. Provisional Application No. 62/052,689, entitled, “Spray DrierAssemblies and Methods For Automated Spray Drying” by Abdul W. Khan etal filed Sep. 19, 2014. The entire teachings of the above application(s)are incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under contractHHS0100201200005C awarded by the Biomedical Advanced Research andDevelopment Authority (BARDA). The Government has certain rights in theinvention.

BACKGROUND

Making up about 55% of the total volume of whole blood, blood plasma isa whole blood component in which blood cells and other constituents ofwhole blood are suspended. Blood plasma further contains a mixture ofover 700 proteins and additional substances that perform functionsnecessary for bodily health, including clotting, protein storage, andelectrolytic balance, amongst others. When extracted from whole blood,blood plasma may be employed to replace bodily fluids, antibodies andclotting factors. Accordingly, blood plasma is extensively used inmedical treatments.

To facilitate storage and transportation of blood plasma until use,plasma is typically preserved by freezing soon after its collection froma donor. Fresh-Frozen Plasma (FFP) is obtained through a series of stepsinvolving centrifugation of whole blood to separate plasma and thenfreezing the collected plasma within less than 8 hours of collecting thewhole blood. In the United States, the American Association of BloodBanks (AABB) standard for storing FFP is up to 12 months from collectionwhen stored at a temperature of −18° C. or below. FFP may also be storedfor up to 7 years from collection if maintained at a temperature of −65°C. or below. In Europe, FFP has a shelf life of only 3 months if storedat temperatures between −18° C. to −25° C., and for up to 36 months ifstored at colder than −25° C. If thawed, European standards dictate thatthe plasma must be transfused immediately or stored at 1° C. to 6° C.and transfused within 24 hours. If stored longer than 24 hours, theplasma must be relabeled for other uses or discarded.

Notably, however, FFP must be kept in a temperature-controlledenvironment of −18° C. or colder throughout its duration of storage toprevent degradation of certain plasma proteins and maintain itsefficacy, which adds to the cost and difficulty of storage andtransport. Furthermore, FFP must be thawed prior to use, resulting in adelay of 30-80 minutes before it may be used after removal from coldstorage.

Accordingly, there is a need to develop alternative techniques theprocessing and storage of plasma.

SUMMARY

A long-standing need and challenge to the blood industry has been toprovide safe, reliable and convenient blood products while preservingthe efficacy and safety of those products in storage and when used intransfusion or as a source for medical treatments. The present inventionprovides efficacy preservation and includes the preservation of theclotting factors in the plasma in a manner that does not otherwise harmthe plasma or the transfused patient. During spray drying, some bloodplasma proteins degrade to some extent, due to shear stress, surfacestress (e.g., air-liquid interfacial stress), exposure to extreme pH,thermal stress, dehydration stress, and other environmental stresses.

The methods and compositions of the present invention recognize that pHand associated stresses can be reduced or the effects of which can beameliorated by the use of novel formulations of the liquid plasma priorto or contemporaneously with spray drying. Formulation of the liquidplasma by citric acid or a similar spray dry stable acidic substance(SDSAS), at novel concentrations, maintains the pH of the plasma at anon-alkaline level during the spray drying process. This results inhigher recovery and better subsequent storage stability of active plasmaproteins when compared to unformulated plasma. FIGS. 4A-C show how theSDSAS of the present invention may be added (formulated)contemporaneously with the plasma in the spray drying process.

The term “recovery” is defined herein as referring to the percentage ofan analyte preserved after spray drying compared with the analyte in asample of the same native plasma (the same sample before spray drying);the analyte is analyzed on native plasma and rehydrated plasma at thesame protein concentrations. The analyte can be any known plasmsubstance such as a protein (e.g., vWF antigen or fibrinogen) and can bemeasured by concentration or activity of the analyte (e.g., vWF:RCoactivity).

A spray dry stable acidic substance (SDSAS) as used herein is anysubstance such as an acid or acidic salt or other substance thateffectuates pH and is physiologically suitable for addition to theplasma being spray dried and physiologically suitable to the subjects(human or otherwise) to which the reconstituted plasma is to beadministered (transfused). The SDSAS remains sufficiently stable (e.g.,does not materially evaporate or chemically breakdown) during the spraydrying process. The SDSAS effectuates the pH adjustment described hereinwhich results, for example, in improved von Willebrand's factor recoveryin the reconstituted plasma described herein. Specific examples known tothe inventors of spray dry stable acidic substances include citric acid,lactic acid, monosodium citrate, glycine HCL and other SDSAS's describedherein. Other SDSAS's may be known in the art or may be determinable bystraightforward experimentation.

Accordingly, spray drying formulation, i.e., treatment of feed plasmaprior to or contemporaneously with spray drying, preserves and allowsrecovery of active clotting factors of rehydrated plasma that hasundergone the spray drying process as well as long term stability duringstorage after drying. As further discussed below, these improvements tocertain embodiments of spray drying of blood plasma involvingformulation with a SDSAS, also improve the ease and lower the cost ofrehydration of the plasma product by allowing the spray dried plasma tobe rehydrated with sterile water (e.g., water for injection: WFI).

The present invention contemplates a method of producing spray driedplasma with improved recovery of active plasma proteins and long termstability of plasma proteins. In an embodiment, the method provides forplasma to be dried, the plasma may be selected from citrate phosphatedextrose solution (CPD) plasma or whole blood (WB) plasma. The methodfurther provides for a SDSAS and a spray drying system. The inventionfurther contemplates adjusting the pH of the CPD plasma or WB plasmawith the SDSAS by bringing the concentration of the acidic compound toabout 0.001 to about 0.050 mmol/mL, which lowers the pH of the plasma toabout 5.5 to about 6.5 or to about 7.2 to create formulated plasma.

The present invention further contemplates drying the formulated plasmawith the spray drying system to create spray dried formulated plasma,said spray dried formulated plasma having a recovery of active vonWillebrand factor (vWF) at least 10 to at least 20 percentage pointsgreater than the recovery of active von Willebrand factor obtained froman otherwise identical spray dried plasma that has not undergone acidformulation with an SDSAS. The SDSAS may be selected from any known inthe art, however, citric acid and lactic acid are preferred substancesfor use in the present invention. The physiologically compatible SDSASis added to the plasma before spray drying and preferable shortly beforespray drying or contemporaneously with spray drying. Additionally, thepH of the plasma may be determined before the addition of a SDSAS to theplasma to determine an appropriate amount of acid to add. In anembodiment, about 7.4 mM of citric acid is added to the CPD plasma or WBplasma. In an embodiment, the pH of the formulated plasma is about 5.5to about 6.5 or to about 7.2. The present invention further contemplatesthat the recovery of vWF may be from about 10 to about 20 percentagepoints to about 40 percentage points greater than the recovery of activevon Willebrand factor obtained from an otherwise identical spray driedplasma that has not undergone pretreatment with a SDSAS or about 26percentage points to about 35 percentage points greater than therecovery of active von Willebrand factor obtained from an otherwiseidentical spray dried plasma that has not undergone pretreatment with aSDSAS.

The present invention contemplates reconstituting the spray driedformulated plasma of the present invention. The spray dried formulatedplasma of the present invention may be reconstituted with anyphysiologically compatible solution. Further, the spray dried formulatedplasma of the present invention may be reconstituted with sterile water(e.g., water for injection (WFI) or similar) or clean, non-sterile waterand, if desired, filtered after reconstitution. It is contemplated thatthe reconstituted spray dried formulated plasma of the present inventionhas a pH of about 6.8 to about 7.6, or about 6.9 to about 7.5.

In an embodiment, a subject in need of plasma is selected and thereconstituted plasma of the present invention is administered to thesubject in need of plasma. Said administration is intravenous.

In an embodiment, it is contemplated that the spray dried formulatedplasma is substantially more stable when stored under refrigeration, atambient temperature or higher temperature, e.g., 37° C., e.g., for twoweeks (see, FIGS. 8 and 9) before reconstitution than the spray driedplasma produced from unformulated liquid plasma. It is furthercontemplated that the stability of the spray dried treated plasma isdetermined by measuring the activity of von Willebrand factor and/orother plasma proteins.

The present invention contemplates a reconstituted spray dried plasmaproduct for human transfusion (administration), the reconstituted spraydried plasma product having been reconstituted with, for example,sterile water and the reconstituted spray dried plasma product having apH of about 6.8 to about 7.6 or about 6.9 to 7.5 and comprising activevon Willebrand factor of greater than 5 percentage points as compared tothe recovery of active von Willebrand factor obtained from an otherwiseidentical spray dried plasma that has not undergone formulation with aSDSAS; or about 5 percentage points to about 40 percentage pointsgreater than the recovery of active von Willebrand factor obtained froman otherwise identical spray dried plasma that has not undergonepretreatment with a SDSAS. The present invention further contemplatesthat the active von Willebrand factor is of about 25 percentage pointsto about 35 percentage points greater than the recovery of active vonWillebrand factor obtained from an otherwise identical spray driedplasma that has not undergone formulation with a non-volatile,physiologically compatible acid.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following more particular description of theembodiments, as illustrated in the companying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of the embodiments.

FIG. 1A is a schematic illustration of an embodiment of a spray dryersystem of the present disclosure, including a spray dryer device 102 anda spray dryer assembly.

FIG. 1B is a schematic illustration of a plurality of the spray dryersystems of FIG. 1A for use with a pooled liquid source.

FIGS. 2A and 2B are thematic illustrations of the spray dryer assemblyof FIG. 1A.

FIG. 3 is a schematic illustration detailing embodiments of a collectionchamber of the spray dryer assembly of FIGS. 2A-2B.

FIG. 4(A-C) are A) a schematic diagram of spray drying system andpossible stress experienced by protein solution and droplet during spraydrying. A) Also shows how contemporaneous dosing may be performed byfeeding the SDSAS into the plasma prior to feeding the plasma into thespray dryer. B) Also shows how contemporaneous dosing may be performedby feeding the SDSAS into the feeding line after the plasma but beforethe spray dryer. C) Also shows how contemporaneous dosing may beperformed by feeding both the plasma and the SDSAS into the spray headsimultaneously.

FIG. 5 panels A-C are schematic illustrations depictingunfolding/refolding model of the vWF A2 domain and protelolysis byADAMTS13. (A) Cartoon of the vWF A2 domain in its native folded state.(B) The first step of unfolding occurs from the C-terminal end of thevWF A2 domain, influenced by the presence of the vicinal disulphide bond(cysteines depicted by C). Initial unfolding occurs up to, or including,the central b4 sheet in which the scissile bond (YM) is contained. Thisunfolding intermediate step exposes the high-affinity ADAMTS13spacer-binding site. (C) Once the stabilizing effect of thecalcium-binding site (CBS) is overcome this results in the completeunfolding of the vWF A2 domain and the positioning of the ADAMTS13active site for nucleophilic attack of the Y1605-M1606 scissile bond

FIG. 6 is a bar graph showing that formulation of plasma with citricacid stabilizes during spray drying ˜50% von WillebrandFactor:Ristocetin Cofactor (vWF:RCo) activity without any impact ofother coagulation factors. This is done at time zero, time uponcompletion of spray drying. CP indicates Control Plasma; SpDP indicatedSpray-Dried Plasma; PreT indicates plasma formulation with SDSAS.

FIG. 7 is a bar graph showing that formulation of plasma with citricacid confers stability to vWF and all other coagulation factors duringstorage at 4° C.

FIG. 8 is a bar graph showing that pre-treatment of plasma with citricacid confers stability to vWF and all other coagulation factors duringstorage at 25° C.

FIG. 9 is a bar graph showing that formulation of plasma with citricacid confers stability to coagulation factors during storage at 37° C.

FIG. 10 is a photographic image showing that formulation of plasm withcitric acid stabilizes vWF during SpD (spray drying).

FIG. 11A is a line graph showing the results activity (IU/dL) of vWF:RCoactivity for CP/FFP and Fed Plasma under constant plasma feeding rate of10 mL/min, but variable aerosol gas flow rates (0, 5, 10, 15, and 20L/min).

FIG. 11B is a line graph showing pH for CP/FFP and the fed plasma underconstant plasma feeding rate of 10 mL/min, but variable aerosol gas flowrates (0, 5, 10, 15, and 20 L/min).

FIG. 12A is a line graph showing the results activity (IU/dL) of vWF:RCofor CP/FFP and Fed Plasma at Aerosol gas flow rates of 10 L/min; fluid=2ml/min, 10 L/min; fluid=4 ml/min, 10 L/min; fluid=6 ml/min, 10 L/min;fluid=8 ml/min, and 10 L/min; fluid=10 ml/min.

FIG. 12B is a bar graph showing pH for CP/FFP and Fed Plasma at Aerosolgas flow rates of 10 L/min; fluid=2 ml/min, 10 L/min; fluid=4 ml/min, 10L/min; fluid=6 ml/min, 10 L/min; fluid=8 ml/min, and 10 L/min; fluid=10ml/min.

FIG. 13 is a bar graph showing the effect of differentSDSAS-formulations on the vWF:RCo recovery and pH during spray. The pHlevels prior to and post spray were shown on the top of the bar graph.

FIG. 14A-C are bar graphs showing the effect of differentSDSAS-formulations on the vWF:RCo recovery and pH during spray drying.(A) citric acid, (B) lactic acid, and (C) pH. The pH levels prior to andpost spray were shown on the top of the bar graph.

DETAILED DESCRIPTION

Embodiments of the present disclosure are directed to methods andcompositions relating to a spray dried liquid sample. In certainembodiments, the liquid sample is plasma obtain from a blood donor. In apreferred embodiment, the blood donor is human. However, it may beunderstood that the disclosed embodiments may be employed to spray dryany biological mixture of solid particles and/or molecules in acontinuous liquid medium, including, but not limited to, colloids,suspensions and sols (a colloidal suspension of very small particles).

Plasma

Plasma is the fluid that remains after blood has been centrifuged (forexample) to remove cellular materials such as red blood cells, whiteblood cells and platelets. Plasma is generally yellow-colored and clearto opaque. It contains the dissolved constituents of the blood such asproteins (6-8%; e.g., serum albumins, globulins, fibrinogen, etc.),glucose, clotting factors (clotting proteins), electrolytes (Na⁺, Ca²⁺,Mg²⁺, HCO_(s) ⁻, Cl⁻, etc.), hormones, etc. Whole blood (WB) plasma isplasma isolated from whole blood with no added agents exceptanticoagulant(s). Citrate phosphate dextrose (CPD) plasma, as the nameindicates, contains citrate, sodium phosphate and a sugar, usuallydextrose, which are added as anticoagulants. The level of citrate in CPDplasma, derived from whole blood, is about 20-30 mM. Thus, the finalcitrate concentration in the whole blood derived CPD plasma formulatedwith 7.4 mM citric acid will be about 27.4-37.4 mM.

The plasma of the present invention may be dried after pooling orunit-by-unit. Pooling of multiple plasma units has some benefits. Forexample, any shortfall in factor recovery on an equal-volume basis canbe made up by adding volume from the pool to the finished product. Thereare negative features as well. Making up volume from the pool to improvefactor recovery is expensive. Importantly, pooled plasma must beconstantly tested for pathogens as any pathogens entering the pool from,for example, a single donor, runs the risk of harming hundreds orthousands of patients if not detected. Even if detected, pathogencontamination of pooled plasma would render the whole pool valueless.Testing can be obviated by pathogen inactivation of the plasma byirradiation or chemically such as solvent detergent treatment; however,each such treatment adds cost and complexity to pooled plasmaprocessing. In any event, pooled plasma processing is generallyunsuitable to the blood centers and generally only really suitable to anindustrial, mass production environment.

Conversely, unit-by-unit (unit) collection and processing is well-suitedto the blood center environment and eliminates the risk of pooled plasmapathogen contamination by allowing for pre-processing testing forpathogens and tracking of the unit to ensure that each unit leaves theblood center site pathogen free. The inventors have discovered thatefficient and effective preservation and recovery of clotting factors isthe standard by which successful unit blood plasma processing should bemeasured. Such efficiency is also very helpful in the pooled plasmaenvironment as well.

Clotting Factors

There are many blood plasma factors associated with clotting. Themethods and compositions of the present invention include recoveringamounts of active/undenatured fibrinogen, Factor V, Factor VII, FactorIX and vWF from rehydrated plasma that has undergone the spray dryingprocess. Such blood plasma factors are important in patient treatmentespecially after trauma injuries to promote clotting of wounds. Thus,rapid administration of plasma is an important factor contributing topositive clinical outcomes. The spray dried plasma of the presentinvention can be readily reconstituted in a few minutes at the locationof the trauma event without moving the patient and without time delay.Further, the spray dried plasma of the present invention has high levelsof functional proteins that are stable for extended periods of timewithout refrigeration or freezing.

vWF has generally been difficult to recover and has become one indicatorfor preservation of all factors. The present invention includesrecovering amounts of active/undenatured vWF, in an amount in rehydratedspray dried plasma that is at least about 5 percentage points or greater(e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 or greaterpercentage points) as compared to amounts of active/undenatured vWF ofrehydrated spray dried plasma that do not undergo the pre-treatmentsteps of the present invention. The present invention includesrecovering amounts of active/undenatured vWF, in an amount in rehydratedspray dried plasma that is at about 5 percentage points to about 40percentage points or about 10 percentage points to about 35 percentagepoints higher as compared to amounts of active/undenatured vWF ofrehydrated spray dried plasma that do not undergo the formulation stepsof the present invention. vWF activity is typically assayed with anassay called the von Willebrand factor:Ristocetin cofactor [vWF:RCo]assay, as is known to those of skill in the art. The vWF:RCo assaymeasures the ability of a patient's plasma to agglutinate platelets inthe presence of the antibiotic Ristocetin. The rate of Ristocetininduced agglutination is related to the concentration and functionalactivity of the plasma von Willebrand factor. Another assay, the vWFantigen assay, measures the amount of vWF protein present in a sample.

Spray Dry Stable Acidic Substance (SDSAS)

The present invention contemplates the use of a physiologicallycompatible spray dry stable acidic substance (SDSAS) as a formulationagent for plasma prior to being spray dried.

While the present invention is not limited by theory, it is presumed bythe inventors that the SDSAS of the present invention (e.g., citricacid, lactic acid, etc.) exerts its effects because it prevents oralleviates the rising of the pH of the plasma during the spray dryingprocess. Non-limiting examples of suitable acids are citric acid andlactic acid. Other non-limiting examples of suitable acids are ascorbicacid, gluconic acid and glycine hydrochloride (glycine HCl). because CO₂is lost from plasma during spray drying, the reaction generatingbicarbonate and H⁺ from CO₂ and H₂O is shifted away from H⁺, therebyincreasing the pH (i.e., Chatelier's principle). Citric acid addition(or other SDSAS of the present invention) helps offset this change.Therefore, the plasma is formulated with the SDSAS of the presentinvention. Because of the formulation step, vWF activity loss is reducedand/or the amount of undenatured vWF is increased, as compared to spraydried plasma not subjected to the formulations steps of the presentinvention.

Because the physiologically compatible SDSAS of the present invention isincluded in this manner, the inventors further found out that therehydration step can be performed by water alone (e.g., WFI).Alternatively, sodium phosphate or other agents can optionally be addedto the rehydration solution. Further, any other suitable rehydrationfluid as can be determined by one of ordinary skill in the art may beused.

From experiments conducted by the inventors with spray drying, it hasbeen discovered that the von Willebrand factor activity level in plasmadried by spray drying is affected, in part, by the shear forcesgenerated during the aerosolization process (see, Examples, below) andan increase in the pH of the plasma. The present invention shows thatthe utilization of a step wherein the plasma is formulated with a SDSASgreatly improves the recovery and stability of active vWF overconditions wherein the SDSAS is not used as a formulation agent.

A SDSAS is a substance which does not evaporate easily at roomtemperature at atmospheric pressure. Typically, the boiling point of theSDSAS will be greater than about 150° C. at atmospheric pressure.Non-volatile acids that are suitable of use as the SDSAS of the presentinvention include phosphorus-containing acids such as, for example,ortho-phosphoric acid, pyrophosphoric acid, meta-phosphoric acid, polyphosphoric acid, alkyl- and aryl-substituted phosphoric and phosphinicacids, phosphorous acid, and the like, and mixtures thereof. Othernon-volatile acids suitable for use as the SDSAS of the presentinvention include, but are not limited to, ascorbic acid, citric acid,lactic acid, gluconic acid, oxalic acid, halogenated acetic acids, arenasulfonic acids, molybdic acid, phosphotungstic acid, tungstic add,chromic acid, sulfamic acid, and the like.

SDSAS useful in the process of the invention are capable of replacingthe volatile acid, i.e. CO₂ that escapes from the plasma during spraydrying. As indicated above, examples or suitable acids include, but arenot limited to, ascorbic acid, citric acid, gluconic acid, and lacticacid.

A volatile acid as defined herein has a pka less than about 3 and aboiling point less than about 150° C. at atmospheric pressure.Typically, the pka of the volatile acid is within the range of about 1to about 15. Non-limiting examples of volatile acids are hydrogenchloride, hydrogen bromide, hydrogen iodide, hydrogen fluoride, aceticacid, formic acid, hydrogen sulfide, hydrogen selenide, sulfur dioxide,fluorosulfonic acid, methane sulfonic acid, trifuoroacetic acid,trifluoromethanesulfonic acid, and the like.

A volatile strong acid can be fixed with an amino acid or like to renderit non-volatile, making it easier to use. For example, volatile hydrogenchloride can be converted to glycine hydrogen chloride (glycine HCl,glycine hydrochloride). Similarly, a corrosive strong acid can beconverted to an acidic salt for use in pretreating plasma prior tospray-drying. Examples include NaHSO₄ and NaH₂PO₄: namely the acidicsalts of sulfuric acid.

Non-volatile acids and acidic salts are collectively defined as andincluded as spray dry stable acidic substance (SDSAS's) in thisinvention.

In an embodiment, the SDSAS of the present invention is added to theplasma within about 30 minutes, about 20 minutes, about 15 minutes,about 10 minutes, about 5 minutes, about 1 minute or time zero (0minutes) of spray drying the plasma. In an embodiment, the SDSAS of thepresent invention is added contemporaneously to the plasma as the plasmais being pumped into the spray drying apparatus. The term“contemporaneously” shall be defined herein as meaning within about 60seconds, about 50 seconds, about 40 seconds, about 30 seconds, about 20seconds, about 10 seconds, about 5 seconds, about 1 second and about 0seconds.

The present inventions relate to adding SDSAS to blood plasma to bespray dried in a time period prior to spray drying short enough toobtain a formulation with the desired pH (“plasma formulation”) and toprevent denaturing or damage of certain plasma protein(s) such as vonWillebrand's factor due to prolonged exposure to the low pH condition.Keeping the time delay to 30 minutes or less between formulation of theplasma with SDSAS and spray drying, as described below, results inimproved recovery of plasma proteins, including von Willebrand factor,without undesirable protein damage due to prolonged exposure to the lowpH condition prior to spray drying.

The time period between acid formulation and spray drying will depend onthe pH/acidity of the plasma formulation created by the mixing of theSDSAS and the plasma. In an embodiment, the time period betweencontacting the SDSAS with the blood plasma and spray drying the plasmais in a range between about 0 seconds (e.g., at the time aeroslizationoccurs: time 0) and about 30 minutes. To minimize protein denaturing,the time between acid formulation of the plasma and spray drying shouldbe kept to minimum. The actual maximum time between formulation andspray drying is determined empirically. This close-in-time formulationat time 0 is referred to herein as “contemporaneous formulation.”

There are a number of methods by which contemporaneous formulation maybe carried out. As illustrated in FIG. 4A, in one embodiment aformulation station is provided in association with the spray dryer. Inconjunction with the formulation station, the weight or volume of thepre-spray dried plasma is determined and an SDSAS dose measured toobtain the desired pH of the plasma formulation. The SDSAS dose may beintroduced into the plasma by any convenient method including byinjection through a port on the plasma bag. A formulation station may bemanually, semi-automatically or automatically operated. Naturally, thetiming of the dosing must be controlled carefully as described above.Timing control may be manual, semi-automatic or automatic.

In another embodiment as shown in FIG. 4B, an appropriate dose of SDSASis introduced into the plasma flow channel of the spray dryer prior tothe spray drying head. SDSAS introduction is controlled manually,semi-automatically or automatically to result in the desired plasmaformulation.

In a further embodiment shown in FIG. 4C, an appropriate dose of SDSASis introduced into the spray drying chamber sufficiently close to thespray drying nozzle so that the SDSAS and plasma are mixed together toform a plasma formulation before spray drying occurs in the spray dryingchamber connected to the spray drying head. SDSAS introduction iscontrolled manually, semi-automatically or automatically to result inthe desired plasma formulation.

Protein Stability

Proteins potentially undergo physical degradation (e.g., unfolding,aggregation, insoluble particulate formation) by a number of mechanisms.Many proteins are structurally unstable in solution and are susceptibleto conformational changes due to various stresses encountered duringpurification, processing and storage. These stresses include temperatureshift, exposure to pH changes and extreme pH, shear stress, surfaceadsorption/interface stress, and so on. Proteins in solutions can beconverted to solid formats (i.e., converted to a powder or other dryformat by having the water and other volatile components of the proteinsolution greatly reduced or removed) for improved storage using a numberof methods.

Freeze drying (also known as lyophilization) is the most commonprocessing method for removing moisture from biopharmaceuticals, and canincrease the stability, temperature tolerance, and shelf life of theseproducts. It is a process wherein a suspension, colloid or solid isfrozen and then “dried” under a vacuum by sublimation (phasetransition). In this process, proteins can suffer from colddenaturation, interface stress [adsorption at the water/ice-interface],exposure to increasing alkaline pH (CO₂ loss), and dehydration stress.Freeze drying is well established within the industry. However, itrequires expensive equipment that takes up a great deal of space withina production facility. Freeze drying also can take days to complete, andmanufacturers that need a powdered product must incorporate agranulation step to the process. In an environment where budgets aretightening, and where time and facility space are at a premium, freezedrying might be a difficult option for some companies. Because of thespace needed, drying plasma by freeze-drying technology is limited toplasma manufacturers, and cannot be implemented in blood centers.

Because of the difficulties inherent with freeze drying of plasma withregard to time, space and cost, the present invention is directedtowards an improved spray drying process for plasma that overcomes theknown difficulties related to the spray drying of plasma.

In the spray-drying process, the viscous liquid is pumped through thefeeding line to the nozzle, where the exiting fluid stream is shatteredinto numerous droplets under aerosol gas. The liquid droplets are metwith dry gas and turned into dry particles. It is a much shorter andless expensive process than the freeze drying process, allowing it to beimplemented in research labs and blood centers. However, in thisprocess, plasma proteins can suffer from extensive shear stress,interface stress, thermal stress, dehydration stress and exposure toextreme pH (see, FIG. 4A).

Aerosolization exposes the liquid sample to shear stress and produces anextremely rapid and very large expansion of the air-liquid interface.The syngerstic effects of shear stress and air-liquid interfacial stresscan cause severe detrimental effects on labile compounds such asproteins. Complex biological molecules are difficult to spray drybecause they are very sensitive to high shear stress. Although somecontrol relating to the amount of shear stress encountered can beobtained by, for example, choice of the type of atomizer used and theaerosolization pressure used, it is very challenging to apply spraydrying technology to human plasma because it contains so many diverseproteins. The diverse proteins may be susceptible to different stressesand this can make it difficult determine processing conditions suitablefor all of the types of proteins found in plasma. In particular, vWF,which is designed by nature to be shear sensitive for its biologicalfunctions, is the most shear-force sensitive human plasma protein. Mostof the other plasma proteins remain largely intact after spray dryingexcept vWF. As shown in the Examples section, spray-drying diminishedvWF activity to below the level of detection (see, Example 1, FIG. 6).

Ionizable amino acid residues have been shown to play important roles inthe binding of proteins to other molecules and in enzyme mechanisms.They also have a large influence on protein structure, stability andsolubility. The types of interactions these side chains will have withtheir environment depend on their protonation state. Because of this,their pKa values and the factors that influence them are a subject ofintense biochemical interest. Strongly altered pKa values are often seenin the active sites of enzymes, to enhance the ability of ionizableresidues to act as nucleophiles, electrophiles or general bases andacids. As a consequence of the change in protonation of these residues,the stability of proteins is pH-dependent. Therefore, we rationalizedthat inhibition of the alkalination of plasma during spray drying canpotentially improve the processing and storage stabilities of manyplasma proteins.

U.S. Pat. No. 8,518,452 (the '452 patent) to Bjornstrup, et al., teachesthe use of citric acid as a pretreatment for lyophilized plasma.

As mentioned above, the spray drying process subjects plasma proteins todifferent forces than are found in the lyophilization process. First,spray drying exposes plasma proteins to high stress forces during theaerolization process as the plasma is forced through the narrow orificeexposed to high rate of air flow that is necessary to create suitablysized droplets for drying. Second, the spray drying process exposesplasma proteins to high temperatures that are necessary to force thewater from the aerosolized droplets. Third, the spray drying processsubjects the plasma proteins to dramatic and rapid increases in pH as aresult of the rapid release of CO₂ during drying. Since lyophilizationdoes not subject plasma proteins to these forces, and especially to thisunique combination of forces, one of ordinary skill in the art would notlook to nor find suggestion or motivation in the lyophilization art withregard to improving the spray drying process for plasma.

Indeed, U.S. Pat. No. 7,931,919 (the '919 patent) to Bakaltcheva, et al.teaches the use of 2 mM citric acid in lyophilized plasms. However,citric acid merely acted as a pH adjuster, did not provide any benefitsfor improving quality of product during acquirement or storage.

The present invention provides for the high recovery rate of vWF and forstorage stability of active plasma proteins; a goal that has eludedthose of skill in the art of drying plasma. In fact, the '452 patentdiscussed above provides no teaching of either recovery or long termstability of active plasma protein function with regard to the disclosedlyophilization process. Further, any specific teething with regard tothe recovery and stability of vWF is missing from the '452 patent. vWFhas been notoriously difficult to recover after the drying of plasma.This lack of teaching in the '452 patent is likely indicative of thefailure of the methods disclosed in the '462 patent with regard tosuccessfully recovering active vWF.

U.S. Pat. No. 7,297,716 (the '716 patent) to Shanbrom teaches the use of2% by weight of citric acid and its salts to reduce bacterial growth andadjust/maintain pH in cryoprecipitates of blood and plasma for enhancingtheir purity and safety. The '716 patent, like the '452 patent providesno teaching of recovered plasma protein activity and stability. Whilethe '716 patent mentions that citrate appears to stabilize labileproteins against heat denaturation, it provides no support for thestatement and provides no teaching with regard to actual recoveredprotein activity or long term stability of recovered proteins,especially vWF.

Thus, the present inventors, in spite of the difficulties associatedwith the spray drying of plasma as known to those of skill in the art,have achieved a spray drying process for plasma that results in highrecovery and high stability of plasma proteins, especially, but notlimited to vWF, wherein the recovery of vWF is in an amount inrehydrated spray dried plasma that is at least about 5 percentage pointsor greater (e.g., about 5, 10, 20, 30, 40, 50, 60, 70, 80 percentagepoints or greater) as compared to amounts of active/undenatured vWF ofrehydrated spray dried plasma that does not undergo the pretreatmentsteps of the present invention.

The compositions and steps of the present invention relate to the impactof the formulation of liquid plasma with a SDSAS, for example, citricacid (CA) on the recovery from the spray drying process and stability(during storage of dried and rehydrated plasma after spray drying) ofvWF and other coagulation factors. This can be done by adding a SDSASsuch as, for example, citric acid or lactic acid to the liquid plasmabefore spray drying begins or contemporaneously with the spray dryingprocess. During the spray drying process, CO₂ loss occurs which causesthe pH of the plasma composition to become more alkaline (e.g., toincrease) and adding SDSAS thereby maintains the plasma pH in a range toprevent significant denaturing of the clotting factors, esp. vWF. Thus,the pretreatment of plasma with citric acid, or other SDSAS, serves atleast three main purposes: 1) increases in-process recovery of plasmaproteins; 2) increases stability of plasma proteins during storage; and3) allows spray dried plasma to be rehydrated with water (e.g., sterilewater, WFI), eliminating the need for a specific rehydration solution.

When liquid plasm is formulated with SDSAS before it is dried, the acidresides in the dried plasma product at a level consistent to improvedstorage lifetime and reduced degradation of clotting factors duringstorage. A “level consistent to improve storage lifetime” also means,herein, at a level that results in a physiological pH uponreconstitution of the spray dried plasma. The use of the SDSAS alsopermits simple rehydration by low cost, readily available water forinjection or, in an emergency, plain water at a physiological pH. Theconvenience, lowered cost and improved safety associated with directrehydration by water is evident. Advantages include savings in beingable to ship dried plasma product without the weight and bulk ofrehydration fluid and savings in the cost from not having to speciallyformulate rehydration fluid and reduction or elimination ofrefrigeration or freezing during storage.

Thus, the inventors have discovered that plasma formulation by a SDSASresults in spray dried plasma that has very high recovery of plasmaproteins, especially vWF, highly improved storage properties of thedried plasma and approximately neutral pH when rehydrated with waterwithout a buffering rehydration fluid. Thus, the present inventionpermits spray dried plasma to be manufactured without the additionalexpense and complexity of pretreatment with additional stabilizers suchas polyols and others known in the art. However, the use of stabilizersis not contraindicated and may be beneficial in some instances.

In a further embodiment, a new composition of matter for blood plasmaspray drying is created by dosing by any means the blood plasma prior tospray drying with added citrate (i.e., citric acid) or other suitableSDSAS at an appropriate concentration, as disclosed herein.

In a further embodiment the newly dosed citrate formulated blood plasmabefore spray drying has a concentration of citrate of about 27.5 mM andabout 40.4 mM, or of about 31.6 mM and 34.2 mM.

In a further embodiment a new spray dried blood plasma product iscreated by spray drying blood plasma formulated with an appropriatelevel of a suitable SDSAS (e.g., citric acid) prior to orcontemporaneously with drying and then drying the blood plasma to thedesired level of moisture. The desired level of moisture is generallybetween 2%-10%, 3%-8% and 4%-6%

In various embodiments, citric acid or other SDSAS is added to theplasma as a formulation. Experiments relating to the effect of citricacid or other SDSAS on protection of the activities of proteins found inplasma are explained further in the exemplification section of thisspecification. The concentrations at which citric acid, for example, isused are between about 1 to about 15 mM, or between about 5 mM to about10 mM (e.g., 7.4 mM). Accordingly, plasma proteins can be preservedbetter when citric acid, at the indicated concentrations, is added to itprior to or contemporaneously with spray drying. The activity of vWF isprovided in the exemplification because this factor is especiallysensitive to denaturing and damage by spray drying (See, FIG. 6 and FIG.7) and, thus, is a good indicator protein to show the beneficial effectsof citric acid or other SDSAS with regard to recovery and stability ofthe spray dried plasma proteins. Examples of other physiologicallycompatible SDSAS are known to those of ordinary skill in the art anddescribed herein.

Spray Dryer and the Spray Drying Process

In general, a spray dryer system (spray dryer device) is provided forspray drying a liquid sample such as blood plasma. In an embodiment, thespray dryer system of the present disclosure includes a spray dryerdevice and a spray dryer assembly. The spray dryer device is adapted, inan aspect, to receive flows of an aerosolizing gas, a drying gas, andplasma liquid from respective sources and coupled with the spray dryerassembly. The spray dryer device may further transmit the receivedaerosolizing gas, drying gas, and plasma to the spray dryer assembly.Spray drying of the plasma is performed in the spray dryer assemblyunder the control of the spray dryer device. Any suitable spray dryingsystem may be used in the present invention. For exemplification, asuitable spray dryer is described below.

In certain embodiments, the spray dryer assembly includes a sterile,hermetically sealed enclosure body and a frame to which the enclosurebody is attached. The frame defines first, second, and third portions ofthe assembly, separated by respective transition zones. A drying gasinlet provided within the first portion of the assembly, adjacent to afirst end of the enclosure body.

A spray drying head is further attached to the frame within thetransition zone between the first and second portions of the assembly.This position also lies within the incipient flow path of the drying gaswithin the assembly. During spray drying, the spray drying head receivesflows of an aerosolizing gas and plasma and aerosolizes the plasma withthe aerosolizing gas to form an aerosolized plasma. Drying gasadditionally passes through the spray drying head to mix with theaerosolized plasma within the second portion of the assembly for drying.In the second portion of the assembly, which functions as a dryingchamber, contact between the aerosolized plasma and the drying gascauses moisture to move from the aerosolized plasma to the drying gas,producing dried plasma and humid drying gas.

In alternative embodiments, the aerosolizing gas may be omitted and thespray dryer assembly head may include an aerosolizer that receives andatomizes the flow of plasma. Examples of the aerosolizer may include,but are not limited to, ultrasonic atomizing transducers, ultrasonichumidified transducers, and piezo-ultrasonic atomizers. Beneficially,such a configuration eliminates the need for an aerosolizing gas,simplifying the design of the spray dryer device and assembly andlowering the cost of the spray dryer system.

The spray drying head in an embodiment is adapted to direct the flow ofdrying gas within the drying chamber. For example, the spray drying headincludes openings separated by fins which receive the flow of drying gasfrom the drying gas inlet. The orientation of the fins allows the dryinggas to be directed in selected flow pathways (e.g., helical).Beneficially, by controlling the flow pathway of the drying gas, thepath length over which the drying gas and aerosolized blood plasma arein contact within the drying chamber is increased, reducing the time todry the plasma.

The dried plasma and humid drying gas subsequently flow into the thirdportion of assembly, which houses a collection chamber. In thecollection chamber, the dried plasma is isolated from the humid dryinggas and collected using a filter. For example, the filter in anembodiment is open on one side to receive the flow of humid air anddried plasma and closed on the remaining sides. The humid drying gaspasses through the filter and is exhausted from the spray dryerassembly.

In alternative embodiments, the filter is adapted to separate thecollection chamber into two parts. The first part of the collectionchamber is contiguous with the drying chamber and receives the flow ofhumid drying gas and dried plasma. The dried plasma is collected in thisfirst part of the collection chamber, while the humid air passes throughthe filter and is exhausted from the spray dryer assembly via an exhaustin fluid communication with the second part of the spray dryer assembly.

After collecting the dried plasma, the collection chamber is separatedfrom the spray dryer assembly and hermetically sealed. In this manner,the sealed collection chamber is used to store the dried plasma untiluse. The collection chamber includes a plurality of ports allowingaddition of water to the collection chamber for reconstitution of theblood plasma end removal of the reconstituted blood plasma for use. Thecollection chamber may further be attached to a sealed vessel containingwater for reconstitution.

When handling transfusion products such as blood plasma, the transfusionproducts must not be exposed to any contaminants during collection,storage, and transfusion. Accordingly, the spray dryer assembly, in anembodiment, is adapted for reversible coupling with the spray dryerdevice. For example, the spray dryer assembly is coupled to the spraydryer device at about the drying gas inlet. Beneficially, so configured,the spray dryer assembly accommodates repeated or single use. Forexample, in one embodiment, the spray dryer assembly and spray dryinghead is formed from autoclavable materials (e.g., antibacterial steels,antibacterial alloys, etc.) that are sterilized prior to each spraydrying operation. In an alternative embodiment, the spray dryer head andspray drying chamber is formed from disposable materials (e.g.,polymers) that are autoclaved prior to each spray drying operation anddisposed of after each spray drying operation.

Reference will now be made to FIG. 1A, which schematically illustratesone embodiment of a spray dryer system 100. The system 100 includes aspray dryer device 102 configured to receive a spray dryer assembly 104.A source of plasma 112, a source of aerosolizing gas 114, and a sourceof drying gas 116 are further in fluid communication with the spraydryer assembly 104. During spray drying operations, a flow of the dryinggas 116A is drawn within the body of the assembly 104. Concurrently, aflow of a blood plasma 112A and a flow of aerosolizing gas 114A are eachdrawn at selected, respective rates, to a spray drying head 104A of theassembly 104. In the spray dryer assembly 104, the flow of blood plasma112A is aerosolized in the spray dryer head 104A and dried in a dryingchamber 104B, producing a dried plasma that is collected and stored forfuture use in a collection chamber 104C. Waste water 122 removed fromthe blood plasma during the drying process is further collected forappropriate disposal.

The spray dryer device 102 further includes a spray dryer computingdevice 124. The spray dryer computing device 124 is adapted to monitorand control a plurality of process parameters of the spray dryingoperation. The spray dryer computing device 124 further includes aplurality of user interfaces. For example, one user interface may allowan operator to input data (e.g. operator information, liquid sampleinformation, dried sample information, etc.), command functions (e.g.,start, stop, etc.). Another user interface may display statusinformation regarding components of the spray drier device (e.g.,operating normally, replace etc.) and/or spray drying processinformation (e.g., ready, in-process, completed, error, etc.).

The spray dryer device 102 is in further communication with a Middlewarecontroller 150. The spray dryer device 102 records one or moreparameters associated with a spray drying operation. Examples of theseparameters includes, but are not limited to, bibliographic informationregarding the blood plasma which is spray dried (e.g., lot number,collection date, volume, etc.), bibliographic information regarding thespray drying operation (e.g., operator, date of spray drying, serialnumber of the spray dryer assembly 104, volume of dried plasma, etc.),process parameters (e.g., flow rates, temperatures, etc.). Uponcompletion of a spray drying operation, the spray dryer device 102communicates with the middleware controller to transmit a selectedportion or all the collected information to the middleware controller150.

For example, a spay drying system 100 may be housed in a blood bankfacility. The blood bank facility receives regular blood donations forstorage. Liquid plasma is separated from whole blood donations, driedusing the spray drying system 100 and subsequently stored until use. Themiddleware controller 160 comprises one or more computing devicesmaintained by the blood bank for tracking stored, dried blood.Beneficially, by providing a spray drying system 100 capable of relayinginformation regarding dried plasma to a middleware controller 150 of theblood center in which it is housed, information regarding the storedblood is then automatically conveyed to the blood center.

In an alternative embodiment, illustrated in FIG. 1B, a plurality ofspray dryer systems 100A, 100B, . . . 100N can be used in combinationwith a pooled plasma source 112′. In general, the pooled plasma source112′ is a bulk source of blood plasma having a volume larger than oneblood unit, as known in the art (e.g., approximately 1 pint or 450 mL).Two or more of the spray dryer systems 100A, 100B . . . 100N can operateconcurrently, each drawing blood for spray drying from the pooled plasmasource 112′, rather than a smaller, local blood source.

The spray dryer systems 100A, 100B . . . 100N in a pooled environmentcan operate under the control of a computing device 124′. The computingdevice 124′ is similar to computing device 124 discussed above, butadapted for concurrent control of each of the spray dryer systems 100A,100B . . . 100N. The spray dryer computing device 124′ furthercommunicates with a remote computing device 150, as also discussedabove.

The use of a pooled plasma source 112′, provides advantages over asmaller, local plasma source, such as plasma source 112. When pooledprior to drying, the pooled liquid plasma can be formulated for pathogeninactivation with UV light, a chemical, and the like. The pooled liquidplasma is dried using one or more spray drying systems 100 of thepresent invention and then the dried plasma can be collect in a singlecollection chamber or a plurality of collection chambers. If the pooledplasma is dried for human transfusion, then each collection containercan be configured with an attached rehydration solution. If the pooledplasma is to be used for fractionation purposes, then it is collected ina configured without the rehydration solution. Further embodiments of aspray dryer device 102 for use with the disclosed spray dryer assembly104 may be found in U.S. patent application Ser. No. 13/952,541, filedon Jul. 26, 2013 and entitled “Automated Spray dryer,” the entirety ofwhich is hereby incorporated by reference.

FIGS. 2A and 2B illustrate embodiments of the spray dryer assembly 104in greater detail. As illustrated in FIG. 2A, the spray dryer assembly104 includes a frame 202. An enclosure or body 204 having first andsecond ends 208A, 208B further extends about and encloses the frame 202.Thus, the body 204 adopts the shape of the frame 202. The enclosure 204may further include a dual layer of film sealed together about theperiphery of the frame 202.

In certain embodiments, the frame 202 may define a first portion 206A, asecond portion 206B, and a third portion 206C of the assembly 104. Thefirst portion of the assembly 206A is positioned adjacent the first end208A of the body 204. The third portion of the assembly 206C ispositioned adjacent to the second end 208B of the enclosure 204. Thesecond portion of the assembly 206B is interposed between the first andthird portions of the assembly 206A, 206C.

The frame 202 further defines first and second transition zones 210A,210B between the first, second, and third portions of the assembly 206A,206B, 206C. For example, the first transition zone 210A may bepositioned between the first and second portions of the assembly 206A,206B and the second transition zone 210B may be positioned between thesecond and third portions of the assembly 206B, 206C. In certainembodiments, the frame 202 may narrow in width, as compared to the widthof the surrounding assembly within the transition zones 210A, and/or210B. The relatively narrow transition zones 210A, 210B help to directthe flow of drying gas 116A through the assembly 104.

In further embodiments, the body 204 may include a drying gas inlet 212,adjacent to the first end 208A. The drying gas inlet 212 may be adaptedto couple with the spray dryer device 102 to form a hermetic and sterileconnection that allows the flow of drying gas 116A to enter the assembly104. In one embodiment, illustrated in FIG. 2A, the drying gas inlet 212is positioned within the first portion of the assembly 206A, at aboutthe terminus of the first end of the body 208A. In this configuration,the flow of drying gas 116A is received within the assembly 104 in adirection approximately parallel to a long axis 250 of the assembly 104.

In an alternative embodiment of the spray dryer assembly 104,illustrated in FIG. 2B, the body 204 may include a drying gas inlet212′. The position of the drying gas inlet 212′ is moved with respect todrying gas inlet 212. For example, the drying gas inlet 212′ may bepositioned within the first portion of the assembly 206A and spaced aselected distance from the terminus of the first end of the enclosure206A. In this configuration, the flow of drying gas 116A may be receivedwithin the assembly 104 in a direction that is not parallel to the longaxis 250 of the assembly 104. For example, in a nom-limiting embodiment,the flow of drying gas 116A is received within the assembly 104 in adirection that is approximately perpendicular to the long axis 250 ofthe assembly 104.

In certain embodiments, the spray dryer assembly 104 may further includea removable cover 218. The cover 218 may be employed prior to couplingof the spray dryer assembly 104 with the spray drier device 102 in orderto inhibit contaminants from entering the spray dryer assembly. Incertain embodiments, the cover 218 may be removed immediately prior tocoupling with the spray dryer device 102 or frangible and penetrated bythe spray dryer device 102 during coupling with the spray dryer assembly104.

The drying gas 116A received by the assembly 104 is urged to travel fromthe first portion 206A, through the second portion 206B, to the thirdportion 206C, where it is removed from the assembly 104. As the dryinggas 116A travels within the first portion of the assembly 206A towardsthe second portion of the assembly 206B, the drying gas 116A passesthrough a first filter 220A which filters the drying gas 116A enteringthe assembly 104 in addition to any filtering taking place within thespray dryer device 102 between the drying gas source 116 and the dryinggas inlet 212. In certain embodiments, the first filter 220A is a 0.2micron filter having a minimum BFE (bacterial filter efficiency) of 10⁶.The filter 220A further helps to ensure the cleanliness of the flow ofdrying gas 116A.

In an embodiment, during primary drying, the flow of drying gas BFEreceived by the spray drier assembly BFE may possess a temperaturebetween about 50° C. and about 150° C. and a flow rate of between about15 CFM to about 35 CFM. The flow of aerosolizing gas 116A can possess aflow rate of between about 5 L/min and about 20 L/min and a temperaturebetween about 15° C. to about 30° C. (e.g., 24° C.). The flow of liquidsample 112A may possess a flow rate of between about 3 ml/min to about20 ml/min. As the plasma is dried, the flow of the aerosolizing gas114A, the flow of drying gas 116C, or both may direct the flow of thedried sample 232 through at least a portion of the spray dryer assembly104 (e.g., the drying chamber, the collection chamber or both).

In an embodiment, the assembly 104 may further include a spray dryinghead 104A, a drying chamber 104B, and a collection chamber 104C in fluidcommunication with one another. The spray drying head 104A may bemounted to the frame 202 and positioned within the first transition zone210A. So positioned, the spray drying head 104A is also positionedwithin the flow of drying gas 116A traveling from the first portion ofthe assembly 206A to the second portion of the assembly 206B. The spraydrying head 104A may be further adapted to receive the flow of plasma112A and the flow of aerosolizing gas 114A through respective feed lines214, 216 and output aerosolized plasma 230 to the drying chamber 104B.

In further embodiments, the drying chamber 104B and collection chamber104C may be positioned within the second and third portions of theassembly 206B, 206C, respectively. The drying chamber 104B inflatesunder the pressure of the flow of drying gas 116A and provides space forthe aerosolized blood plasma 230 and the flow of drying gas 116A tocontact one another. Within the drying chamber 104B, moisture istransferred from the aerosolized blood plasma 230 to the drying gas116A, where the drying gas 116A becomes humid drying gas 234. Theaerosolized flow of blood plasma 230 and the flow of drying gas 116A arefurther separated, within the drying chamber 104B, into dried plasma 232and humid drying gas 234. In certain embodiments, the dried plasma 232may possess a mean diameter of less than or equal to 25 μm.

The humid drying gas 234 and dried plasma 232 are further drawn into thecollect chamber 104C through an inlet port 222A of the collectionchamber 104C, positioned within the second transition zone 210B,connecting the collection chamber 104C and the drying chamber 104B. Thecollection chamber 104 includes a second filter 220B which allowsthrough-passage of the humid drying gas 234 and inhibits through-passageof the dried plasma 232. As a result, the humid drying gas 234 passingthrough the filter 220B is separated from the dried plasma 232 andexhausted from the collection bag 104C through an exhaust port 2228 ofthe collection chamber 104C that forms the second end 208B of the body204. For example, a vacuum source (e.g., a vacuum pump) may be fluidcommunication with the exhaust port 222B of the collection chamber 104Cto urge the humid drying gas 234 through exhaust port b. Concurrently,the dried plasma 232 is retained in a reservoir 228 of the collectionchamber 104C. The collection chamber 104C is subsequently hermeticallysealed at about the inlet and exhaust ports 222A, 222B, and detached(e.g., cut) from the spray dryer assembly 104, allowing the collectionchamber 104C to subsequently function as a storage vessel for the driedplasma 232 until use.

With reference to FIG. 3, the collection chamber 104C further includes aplurality of one-way valves 702A, 702B positioned at about the inletport 222A and the exhaust port 222B, respectively. The one-way valve702A may function to permit gas flow from the drying chamber 104B to thecollection chamber 104C and inhibit gas flow from the collection chamber104C to the drying chamber 104B. The one-way valve 702B may function topermit gas flow from the collection chamber 104C while inhibiting gasflow into the collection chamber 104C via the exhaust port 222B.

The collection chamber 104C may be further configured for use inrehydrating the dried plasma 232. For example, the collection chamber104C may include a rehydration port 224, a plurality of spike ports 226,and a vent port 228. The rehydration port 224 may be used to communicatewith a source of rehydration solution, allowing the rehydration solutionto come in contact with the dried plasma 232 within the collectionchamber 104C to form reconstituted plasma. The reconstituted plasma maybe subsequently drawn from the collection chamber 104C through the spikeports 226.

The discussion will now turn to further embodiments of spray dryingprocesses which include secondary plasma drying operations, as discussedin U.S. patent application Ser. No. 14/670,127, which is incorporatedherein by reference. In brief, it has been recognized that high levelsof residual moisture in stored, dried plasma (e.g., moisture contentsabove about 3% to about 10%, as compared to the moisture content of theliquid plasma) reduce the shelf life of the dried plasma. However, giventhe relatively low moisture content of the dried plasma collected withinthe collection chamber, exposure of this collected, dried plasma toelevated temperatures may result in damage to one or more the plasmaproteins, rendering the dried plasma unsuitable for later reconstitutionand use. Accordingly, embodiments of secondary drying operationsdiscussed herein are designed to complement the primary spray dryingprocesses discussed above, allowing for further reduction in themoisture content of the plasma after primary drying is completed,without significantly damaging the plasma proteins. As a result, thedried plasma stored after undergoing primary and secondary dryingpossesses an improved shelf life, while remaining suitable for laterreconstitution and use. It has been identified that embodiments of thesecondary drying processes discussed in U.S. patent application Ser. No.14/670,127 may be employed to produce dried plasmas having less than orequal to about 3% moisture content, as compared to the liquid plasma,without significant damage to the plasma proteins, when performed attemperatures of less than or equal to but 70° C. Such secondary dryingprocedures are compatible with the invention of the present application.

The entire teachings of the all applications, patents and referencescited herein are incorporated herein by reference. Specifically, U.S.Pat. Nos. 7,993,310, 8,469,202, 8,533,971, 8,407,912, 8,595,950,8,601,712, 8,533,972, 8,434,242, U.S. Patent Publication Nos.2010/0108183, 2011/0142885, 2013/0000774, 2013/0126101, 2014/0083627,2014/0083628, 2014/0088768, and U.S. patent application Ser. No.14/670,127 are incorporated herein by reference and are instructive ofwhat one of ordinary skill in the art would know and understand at thetime of the present invention.

Ranges of values include all values not specifically mentioned. Forexample, a range of “20% or greater” includes all values from 20% to100% including 35%, 41.6%, 67.009%, etc., even though those values arenot specifically mentioned. The range of 20% to 30% shall include, forexample, the values of 21.0% and 28.009%, etc., even though those valuesare not specifically mentioned.

The term “about,” such as “about 20%” or “about pH 7.6,” shall mean ±5%,±10% or ±20% of the value given.

EXEMPLIFICATION Abbreviations and Nomenclature

FFP—Fresh Frozen Plasma manufactured from CPD Whole Blood; plasm notfiltered. Plasma is placed in −18° C. freezer within 8 hours ofcollection.

CP: control plasma, referring to plasma before spray drying

CP/FFP: FFP control plasma

Batch—represents a unique spray drying run at Velico.

SpDP/FFP—Spray dried plasma manufactured from thawed FFP

SpD: spray-drying

SpDP: spray-dried plasma

Feed plasma: liquid plasma to be fed through a feeding tube tospray-drying device

Fed plasma: liquid plasma having been fed to the system without beingsprayed

Sprayed plasma: fed plasma subjected to aerosolization

vWF: von Willebrand factor

vWF:RCo: vWF activity measured by vWF ristocetin assay

PreT: pretreatment or pre-treated (formulation or formulated)

CA: citric acid

PreT/CA: pre-treated (formulated) feed plasma with citric acid

RS-CA: citric acid rehydration solution (3.5 mM citric acid)

RS-CAP: citric acid rehydration solution, buffered with sodium phosphate(pH 3.5)

WFI: water for injection

SDSAS: spray dry stable acidic substance

Example 1: Enhancing In-Process (Spray-Drying) Stability of vWF Factorand Storage Stability of Multiple Plasma Proteins by Treating the FeedPlasma with Citric Acid Prior to Spray Drying

Introduction

von Willebrand factor (vWF) is a large adhesive glycoprotein withestablished functions in hemostasis. It serves as a carrier for factorVII and acts as a vascular damage sensor by attracting platelets tosites of vessel injury. The size of vWF is important for this latterfunction, with larger multimers being more hemostatically active.Functional imbalance in multimer size can variously cause microvascularthrombosis or bleeding. The regulation of vWF multimeric size andplatelet-tethering function is carried out by ADAMTS13, a plasmametalloprotease that is constitutively active. It is secreted into bloodand degrades large vWF multimers, decreasing their activity. Unusually,protease activity of ADAMTS13 is controlled not by natural inhibitorsbut by conformational changes in its substrate, which are induced whenvWF is subject to elevated rheologic shear forces. This transforms vWFfrom a globular to an elongated protein. This conformationaltransformation unfolds the vWF A2 domain and reveals cryptic exosites aswell as the scissile bond. To enable vWF proteolysis, ADAMTS13 makesmultiple interactions that in the protease to the substrate and positionit to engage with the cleavage site as this becomes exposed by shearforces (FIG. 5). ADAMTS 13 (a disintegrin and metalloproteinase with athrombospondin type 1 motif, member 13), also known as von Willebrandfactor-cleaving protease (vWFCP), is a zinc-containing metalloproteaseenzyme.

During spray drying (SpD), the plasma proteins are subject toconsiderable shear forces due to the spraying mechanism as the solutionsare fluidized through a fine nozzle to form the droplets in contact withdrying air. FIG. 4a is a schematic diagram showing the various shearforces proteins are subject to during spray drying. The process ofunfurling multimeric vWF is expected to be triggered by the hydrodynamicforces of elevated shear stress during SpD in combination withair-liquid interface stress. The shear-induced structural change of vWF,when combined with other physical factors associated with SpD, such ashigh temperature and/or unfavorable pH as well as the air-liquidinterface stress, may lead to protein denaturation (if unfolded vWFfails to refold properly post-SpD) and proteolytic degradation (unfoldedvWF exposes proteolytic sites for ADMATS13), impairing the vWF activityin the spray dried plasma (SpDP), as well as other proteins.

Spray drying can be optimized to reduce the protein damage caused byshear force and temperature through mechanical engineering. However, thepH rise is inevitable during SpD due to the loss of CO₂, driven by bothspraying and drying sub-processes. Further, the elevated pH isparticularly undesirable for SpDP during storage. SpDP contains aresidual amount of water and an alkaline pH will accelerate proteindegradation during storage. Therefore, it is highly desirable tomaintain the physiological pH during and post SpD. This can be done byadding a non-volatile spray dry stable acidic substance (SDSAS),preferably a physiologically compatible weak acid such as citric acid orlactic acid, to the liquid plasma to counterbalance the CO₂ loss byinhibiting pH rise during SpD and thereby allow SpDP to be stored, at anon-alkaline pH. In summary, pretreatment or contemporaneous treatmentof plasma with citric acid serves three main purposes: 1) it increasesin-process stability of plasma proteins; 2) it increases stability ofplasma proteins during storage; and 3) it allows SpDP to be rehydratedwith water, eliminating the need for a rehydration solution.

Objectives

The object of this study is to evaluate the impact of a SDSASformulation of plasma with citric acid on the recovery from SpD andstability during storage of SpDP of vWF and other coagulation factors inSpDP.

Study Design and Methods

Plasma samples were formulated by the addition of citric acid from a 20%stock solution prior to spray drying. Plasma samples were spray driedusing a drying gas inlet temperature of 125° C., plasma fluid rate of 10ml/min, aerosol gas rate of 20 L/min and the exhaust temperature wasmaintained at 55° C. The clotting factors fibrinogen, Factors V, VII,VIII and IX, von Willebrand factor (vWF), prothrombin time (PT) andactivated partial prothromboblastin time (aPTT) were determined afterspray drying and after storage at 37° C. room temperature andrefrigeration. vWF multimer analysis was carried out at the Blood Centerof Wisconsin (BCW) as follows. Plasma samples, loaded at equal vWF:Aglevels (0.2 mU), were analyzed by 0.65% LiDS-agarose gel electrophoresisand western blotting with chemiluminescent detection using the FujifilmLAS-300 luminescent image analyzer. Densitometry was performed andarea-under-the curve calculated. The percentage of low (L), intermediate(I) and high (H) molecular weight (MW) multimers (M) were calculated.Formulated SpDP samples were rehydrated with water for injection (WFI),standard SpDP samples (i.e., control samples without added pretreatmentagents as listed here) were rehydrated in Citrate-Phosphate Buffer(CPB).

Results

As shown in FIG. 6, SpD resulted in a loss of coagulation factoractivity between 0% and up to 20% (FV, FVII, FVIII and FIX), but had noimpact on fibrinogen and vWF antigen levels. However, it lowered thevWF:RCo activity below detection, which, remarkably, increased recoveryby 50% by formulation. Consistent with the excellent recoveries of thecoagulation factors and fibrinogen, SpD had no adverse effect on PT. SpDslightly prolonged aPTT (comparing Bar 1 and 3 in the aPTT cluster).Citric acid formulation prolonged aPTT of the plasma even before SpD,suggesting that interference of added citric acid in the assay, likelyby taking some free calcium required by multi-steps in the intrinsicpathway, collectively measured as aPTT when combined with the commonpathway. However, SpD had no impact on aPTT of the formulated plasma(comparing Bar 2 and 4 in aPTT cluster).

When stored refrigerated for 6 weeks, coagulation factors in the plasmasamples did not lose more than 10% of their activities (FIG. 7.).However, the benefits of Pre-T/CA were highlighted after 2 weeks at 25°C. (FIG. 8) and even more so at 37° C. (FIG. 9). All characterizedparameters performed better for Pre-T/CA SpDP than standard SpDP.

To gain insight into steep decline, and dramatic salvage of vWF:RCoactivity by plasma formulation, vWF multimer quantifications wereperformed by the inventors on plasma samples pre and post-SpD, with orwithout pretreatment. The results are shown in FIG. 10. Positive andnegative controls were also included. As rationalized in theintroduction to the example, SpD took a heavy toll on vWF multimers,almost completely depleted high molecular weight vWF multimers (HMWM),which was paralleled by an increase in low molecular weight multimers(LMWM). However, Pre-T/CA greatly increased recovery of HMWM multimers,consistent with vWF:RCo data. Lane 13: Type 28 vWF Control=Type 2B vonWillebrand disease. Lane 14: Healthy Control. Lane 15: CP=Controlplasma. Lane 16: CP/PreT=control plasma plus citric acid. Lane 17:SpDP=reconstituted spray dried plasma. Lane 18: SpDP/PreT=reconstitutedspray dried plasma power formulated with citric acid.

Conclusions

Surprisingly, SpD exerts a heavy toll on vWF multimer formation andactivity. The results show that vWF is sensitive to shear stress whichadversely affects its size and biological function. Shear stressenhances the proteolysis of vWF in normal plasma. Presumably, and whilenot limiting the present invention to theory, the synergistic effects ofshear force during aerosolization, pH change and thermal stress, causesunfolding of vWF. Formulation of plasma with a SDSAS greatly improvesthe recovery of shear force labile vWF, increases the stability ofmultiple plasma proteins during storage and simplifies rehydration. SpDPsubjected to formulation showed improved profiles of PT, fibrinogen, FV,FVII, FVIII, FIX and vWF antigen (Ag) levels when stored 2 weeks and 4°C. and 25° C.

Example 2: Characterization of the Effect of Aerosol Flow Rate on vWFFactor

Background

The spray-drying process can be divided into feeding, spraying, anddrying stages. Each sub-process can potentially cause damage to plasmaproteins, especially vWF (FIG. 4). Identification of the criticalstep(s) to vWF degradation can aid in process development minimizingprocessing damage to plasma proteins. In this example, the impact ofspraying on vWF recovery was evaluated.

Study Design and Methods

Thawed FFP samples were fed at 10 mL/minute under variable aerosol gasflow (0, 5, 10, 15 or 20 L/minute) without drying gas on. Thesesettings, allowing the plasma to be fed into the system, with or withoutaerosolization in the absence of heating, allowed study of the impact ofplasma feeding and spray/aerosol gas flow rate in the spray-dryingprocess. The sprayed liquid plasma samples were analyzed for pH andvWF:RCo.

Results

The results are shown in FIGS. 11A and 11B. Plasma feeding at 10 mL/minwithout aerosol gas flow (0 L/min) allowed the evaluation of the impactof feeding alone on vWF recovery. Plasma feeding alone had nosignificant impact on either pH or vWF.

vWF still remained intact at 5 L/min of aerosol gas flow, but the pH wassharply elevated to approximately 8.0 (FIG. 11B). However, increase ofthe aerosol gas flow to 10 L/min eliminated 50% vWF:RCo activity, andsuffered more damage as aerosol gas flow increased to 15 and 20 L/min(FIG. 11A). The pH remained at about 8 as the aerosol gas flow wasincreased from 5 to 20 L/min, indicating near complete loss of CO₂ inthe plasma upon aerosolization. The lack of correlation between pH riseand vWF:RCo activity at 5 L/min suggests that transient exposure toslight alkaline pH (8.0) alone did not cause detectible damage to vWF.

Escalation of aerosol gas flow downsizes the plasma droplets, which hasmultiple consequences. The reduced droplet size increased exposure ofplasma proteins to air/liquid interfacial stress. The combination ofelevated aerosol gas flow and reduced droplet size increased speed ofthe droplet motion in the gas, thereby aggravating the shear stress toproteins on the droplet surface, which have already been stressed frominteraction with the air/liquid interface.

Conclusion

This study firmly established the correlation between aerosolization andvWF factor deterioration.

Example 3: Characterization of the Effect of Plasa Feeding Rate on vWF

Background

Example 2 identified the spray sub-process as a major stress factorresponsible for vWF degradation during spray drying. This indicates thatthe critical negative contribution of the combined shear and air/liquidinterfacial stresses was exerted on the plasma droplets (and,consequently, on the plasma proteins) while traveling at a high rate ofspeed upon aerosolization. It also suggested that the impact of thecombined shear and air/liquid interfacial stresses on plasma proteinsupon aerosolization can be further modified by altering the dropletsize. Droplet size can be modified by varying the plasma feed rate undera constant aerosol flow rate. In this example, plasma was fed into thesystem at different rates under constant aerosol flow rate. Largerdroplets at a higher plasma feeding rate would have less air-liquidinterface exposure for plasma proteins and have slower motion rate andlower shear stress for plasma proteins. Thus, the plasma proteins willsustain less stress attributed to air-liquid interface force and shearforce.

Study Design and Method

Thawed FFP samples were fed at 2, 4, 6, 8 or 10 mL/min under a constantaerosol gas flow of 10 L/min without drying gas on. The sprayed liquidplasma samples were analyzed for vWF:RCo activity and pH.

Results

Consistent the observations in Example 2, at 10 L/min of aerosol gasflow, vWF:RCo activity dramatically declined after spraying between 2and 10 mL/min of plasma input (FIG. 12). vWF:RCo recovery trendedslightly higher as plasma input rate increased from 2 to 10 mL/min. pHwas significantly increased under all conditions, trending lower from pH8.3 at 2 mL/min to 7.9 at 10 mL/min as the plasma feed rate increased(FIG. 12B). The opposite trends for pH and vWF:RCo with respect toplasma feeding rate are consistent with the increase of droplet sizes asthe result of the increase plasma feeding rate. This reduced theair/liquid-interface to mass ratio and, consequently, the shear andair/liquid-interface stresses as well as CO₂ loss.

Conclusion

The results further established the inverse relationship between vWFrecovery and spray stresses.

Example 4. The Effect of Formulation of Plasma with Different Spray DryStable Acidic Substance (SDSAS's) on vWF Recovery during Spray

Background

Example 1 highlighted the importance of controlling the pH of the feedplasma in reducing the detrimental effect of spray-drying on vWF.Examples 2 and 3 identified the spray sub-process as a critical stepleading to the degradation of vWF. Taken together, these data suggestthat reducing the destructive effect of spray on vWF by lowering the pHof feed plasma is critical for improving the overall quality of SpDP. Inthis example, the impact of pretreatment on the preservation of vWFfactor during spray was explored using a diverse panel of SDSAS's.

Study Design and Methods

Aliquots of thawed FFP were formulated separately with a wide range ofSDSAS's including ascorbic acid, citric acid, gluconic acid, glycinehydrogen chloride (glycine-HCl), lactic acid and monosodium citrate. Theamount of the treating chemical was pre-determined by titrating theunformulated SpDP rehydrated with WFI to ˜pH 7.3. Control plasma includeformulated and hyper-formulated (7.4 mM citric acid in Example 1) plasmasamples.

Results

The results are shown in FIG. 13. Spraying of the naïve plasma led to asharp rise in pH (pH 7.3 and 8.0 before and after spraying,respectively; 7.3/8.0, Bar 2) and reduced vWF:RCo activity by about 70%(30% recovery) (Bar 2). Formulation of the plasma with 7.4 mM citricacid, which lowered the pH to 6.3 in the feed plasma and resulted in alower than the physiological pH after spraying (6.9), reduced by about50% vWF:RCo activity during spraying (50% recovery) (Bar 3). Formulationwith 7.4 mM monosodium citrate, which lowered the pH to 6.7 in the feedplasma and resulted in a physiological pH after spraying lowered vWF:RCoactivity recovery by about 40% (Bar 4), which was higher than naïveplasma (Bar 2). Formulation with other SDSAS's, citric acid (4.7 mM, Bar5), ascorbic acid (Bar 6), glycine HCl (Bar 7), gluconic acid (Bar 8)and lactic acid (Bar 9), all of which lowered the plasma pH to ˜6.7 andresulted in a physiological pH (˜7.3) after spraying, led to similarvWF:RCo activity recovery of about 40% after spray. Taken together,these results indicated that lowering the pH of feed plasma is criticalfor preserving vWF during spray.

Conclusion

Enhanced vWF preservation can be achieved by formulating the feed plasmawith a wide array of SDSAS's—not only citric acid, but monosodiumcitrate, ascorbic acid, glycine HCl, gluconic acid and lactic acid, andprobably many others meeting the criteria given in the presentspecification. However, the most important consideration in choosing theproper SDSAS is the suitability for transfusion. Other important factorsinclude availability of USP grade formulation, tolerance for terminalsterilization and interference with standard assays, to name a few. Asplasma already contains citric acid (as an anticoagulant), addition ofmore citric acid to bring the concentration identified in the presentinvention as being suitable for enhanced plasma protein recovery andstability has the advantage of not introducing a new component to serveas a pH adjuster. Further, citrate is usually rapidly metabolized by theliver. However, rapid administration of large quantities of stored bloodmay cause hypocalcaemia and hypomagnesaemia when citrate binds calciumand magnesium. This can result in myocardial depression or coagulopathy.Patients most at risk are those with liver dysfunction or neonates withimmature liver function having rapid large volume transfusion. Slowingor temporarily stopping the transfusion allows citrate to bemetabolized. Administration of calcium chloride or calcium gluconateintravenously into another vein can be used in order to minimize citratetoxicity. Nevertheless, the elevation of citrate in SpDP can be avoidedby using alternative SDSAS's such as lactic acid and glycine-HCl. Lacticacid is an important constituent in Ringer's Lactate solution, which isoften used for fluid resuscitation after a blood loss due to trauma,surgery, or a burn injury. GlycineHCL is referenced in the USPharmacopeia.

Example 5: Enhanced vWF Factor Protection during Spray is InverselyCorrelated with the pH Levels of the Feed Plasma

Background

Results from Example 4, evaluating different chemicals for lowering thepH of the feed plasma, confirmed the generality of the inhibition of pHrise during spray improves vWF:RCo activity recovery. However, it isstill striking that vWF factor is better preserved at an acidic pH lowerthan the physiological pH (7.2-7.4) during the spraying process.Nevertheless, the surprising observation suggested the potential of pHmanipulation for further improving vWF factor recovery. In this example,we further evaluated pH of the feed plasma with regard to vWF:RCoactivity recovery after spraying. Citric acid and lactic acid werechosen for use in the study.

Study Design and Method

Aliquots of thawed FFP were formulated with different concentrations ofcitric acid or lactic acid from 20× stock solutions. The amount of theformulation chemicals was pre-determined ensuing a physiological orlower pH level of SpDP when rehydrated with WFI. The formulated sampleswere determined for pH, sprayed, and the recovered liquid samples wereanalyzed for pH and vWF:RCo activity.

Result

The results are shown in FIG. 14A for citric acid and FIG. 14B forlactic acid. Consistent with earlier observations, spraying alone led toa rise in pH (not shown) and vWF:RCo deterioration under all conditions.Remarkably, vWF:RCo recovery trended higher as the concentration ofcitric acid or lactic acid increased or pH declined. The inversecorrelation between pH of the feed plasma and vWF:RCo activity recoverywas clearly shown in FIG. 14C, which was generated by pooling data ofboth citric acid and lactic acid studies.

Conclusion

Feed plasma pH can be further exploited to increase vWF recovery inconjunction with recover of other plasma proteins.

What is claimed is: 1) A reconstituted dried plasma product for humantransfusion made by the method of: a) combining i) plasma with ii) oneor more stable acidic substances, wherein said one or more stable acidicsubstances is an acid or acidic salt that effectuates a pH and isphysiologically suitable for addition to plasma being dried orphysiologically suitable for subjects into which reconstituted plasma istransfused, to thereby create a formulated plasma so that the pH offormulated plasma is between about 5.5 and 7.2, b) drying the formulatedplasma to thereby obtain a dried formulated plasma; and c)reconstituting said dried formulated plasma with sterile water oralkaline solution to thereby obtain a reconstituted dried plasma productfor human transfusion. 2) The reconstituted dried plasma product ofclaim 1, said reconstituted plasma has a pH of about 6.8 to about 7.6.3) The reconstituted dried plasma product of claim 1, wherein whencombining the plasma with the one or more stable acidic substances, saidpH of said plasma is adjusted with said one or more stable acidicsubstance up to 30 minutes before drying. 4) The reconstituted driedplasma product of claim 1, wherein when combining the plasma with theone or more stable acidic substances, said pH of said plasma is adjustedby adding one or more stable acidic substances immediately prior to orcontemporaneously with drying. 5) The reconstituted dried plasma productof claim 1, wherein the pH of the plasma is known prior to addition ofsaid one or more stable acidic substances and the amount of said one ormore stable acidic substances to be added plasma is determined based onthe known pH of said plasma. 6) The reconstituted dried plasma productof claim 1, wherein said plasma comprises CPD (citrate phosphatedextrose solution) plasma or is WB (whole blood) plasma. 7) Thereconstituted dried plasma product of claim 1, wherein the one or morestable acidic substances is selected from the group consisting ofascorbic acid. citric acid. gluconic acid, lactic acid, glycinehydrochloride, monosodium citrate, oxalic acid, halogenated aceticacids, arene sulfonic acids, molybdic acid, phosphotungstic acid,tungstic acid, chromic acid, sulfamic acid and any combination thereof.8) The reconstituted dried plasma product of claim 1, wherein citricacid is added to the plasma to increase citrate concentration by 7.4 mM.9) The reconstituted dried plasma product of claim 1, wherein saidformulated plasma has a pH of about 5.5 to about 6.5. 10) Thereconstituted dried plasma product of claim 1, wherein von Willebrandfactor activity from reconstituted plasma is between about 5 and about40 percentage points greater than the von Willebrand factor activityobtained from an otherwise identical dried plasma that has not undergoneformulation with one or more stable acidic substances. 11) Thereconstituted dried plasma product of claim 8, wherein von Willebrandfactor activity from reconstituted plasma is between about 10 and about35 percentage points greater than the von Willebrand factor activityobtained from an otherwise identical dried plasma that has not undergoneformulated with one or more stable acidic substances. 12) Thereconstituted dried plasma product of claim 1, wherein said driedformulated plasma is stable at ambient temperature for at least 7 days.13) The reconstituted dried plasma product of claim 1, furthercomprising measuring activity of Factors V, VII, VIII and IX, or anycombination thereof to determine stability of the reconstituted plasma.14) The reconstituted dried plasma product of claim 1, wherein thereconstituted plasma has a physiological pH. 15) The reconstituted driedplasma product of claim 1, wherein the reconstitution solution comprisesa substance selected from the group consisting of: sterile water, sodiumbicarbonate, disodium phosphate, and glycine sodium hydroxide. 16) Adried plasma product for human transfusion made by the method of: a)combining i) plasma with ii) one or more stable acidic substances,wherein said one or more stable acidic substances is an acid or acidicsalt that effectuates a pH and is physiologically suitable for additionto plasma being dried or physiologically suitable for subjects intowhich reconstituted plasma is transfused, to thereby create a formulatedplasma so that the pH of formulated plasma is between about 5.5 and 7.2,and b) drying the formulated plasma to thereby obtain a dried formulatedplasma. 17) The dried plasma product of claim 16, wherein when saidformulated plasma is dried and reconstituted to obtain reconstitutedplasma, said reconstituted plasma has a pH of about 6.8 to about 7.6.18) The dried plasma product of claim 16, wherein when combining theplasma with the one or more stable acidic substances, said pH of saidplasma is adjusted with said one or more stable acidic substance up to30 minutes before drying. 19) The dried plasma product of claim 16,wherein when combining the plasma with the one or more stable acidicsubstances, said pH of said plasma is adjusted by adding one or morestable acidic substances immediately prior to or contemporaneously withdrying. 20) The dried plasma product of claim 16, wherein the pH of theplasma is known prior to addition of said one or more stable acidicsubstances and the amount of said one or more stable acidic substancesto be added plasma is determined based on the known pH of said plasma.21) The dried plasma product of claim 16, wherein said plasma comprisesCPD (citrate phosphate dextrose solution) plasma or is WB (whole blood)plasma. 22) The dried plasma product of claim 16, wherein the one ormore stable acidic substances is selected from the group consisting ofascorbic acid, citric acid, gluconic acid, lactic acid, glycinehydrochloride, monosodium citrate, oxalic acid, halogenated aceticacids, arctic sulfonic acids, molybdic acid, phosphotungstic acid,tungstic acid, chromic acid, sulfamic acid and any combination thereof.23) The dried plasma product of claim 16, wherein citric acid is addedto the plasma to increase citrate concentration by 7.4 mM. 24) The driedplasma product of claim 16, wherein said formulated plasma has a pH ofabout 5.5 to about 6.5. 25) The dried plasma product of claim 16,wherein when the formulated plasma is reconstituted to obtainreconstituted plasma, von Willebrand factor activity from reconstitutedplasma is between about 5 and about 40 percentage points greater thanthe von Willebrand factor activity obtained from an otherwise identicaldried plasma that has not undergone formulation with one or more stableacidic substances. 26) The dried plasma product of claim 25, wherein vonWillebrand factor activity from reconstituted plasma is between about 10and about 35 percentage points greater than the von Willebrand factoractivity obtained from an otherwise identical dried plasma that has notundergone formulated with one or more stable acidic substances. 27) Thedried plasma product of claim 16, wherein said dried formulated plasmais stable at ambient temperature for at least 7 days. 28) The driedplasma product of claim 16, further comprising measuring activity ofFactors V, VII, VIII and IX or any combination thereof to determinestability of the reconstituted plasma. 29) A reconstituted spray driedplasma product for human transfusion, the reconstituted spray driedplasma product having been formulated with one or more spray dry stableacidic substances (SDSAS), wherein said one or more SDSAS is an acid oracidic salt that effectuates a pH and is physiologically suitable foraddition to plasma being spray dried or physiologically suitable forsubjects into which reconstituted plasma is transfused and having beenreconstituted with sterile water. the reconstituted spray dried plasmaproduct having a of about 6.8 to about 7.6. 30) A spray dried plasmaproduct for reconstitution and transfusion into a human, the spray driedplasma product having been formulated with one or more spray dry stableacidic substances (SDSAS), wherein said one or more SDSAS is an acid oracidic salt that effectuates a pH and is physiologically suitable foraddition to plasma being spray dried or physiologically suitable forsubjects, wherein that the pH of formulated plasma is between about 5.5and 7.2.